Hi I am trying to find sRNA in bacteria using RNA-seq, I know that it's possible find them by structure conservation or sequence homology.but I don't know how to work with RNA-seq data to find them. if anyone can help me I would appreciate.
Your question is a bit wide but I'll try to give you some answers.
First of all, it's not so trivial to include the sRNAs in your RNA-seq library, most protocols select for size and omit short RNAs, if you'll see tRNAs or known sRNAs in your data it's a good sign, if you can't find them you have to adjust the library construction protocol for short RNAs.
The first step would be to map the reads to the genome (if you have a reference) and then look for short regions with high coverage, if your sequencing is paired-end you can see the exact termination point of the transcript - look for a rho independent terminator (GC rich followed by poly-U tract). You should look in intergenic regions although they are present everywhere including in coding sequences and genes UTRs.
I you work on E. coli or Salmonella then there are some papers that found novel sRNAs using Hfq (sRNAs chaperon) RIP-seq.
Hi thanks for reply and sorry excuse my tardiness. I dont understand exactly to what do you refer about tRNA presence, see tRNA where, when i detect some high coverage regions?. Additionaly i'm looking for a workflow or pipeline "tested" to find them, because now i'm doing something similar but i don't know if what im doing is right.
Map against a reference genome --> get high coverage RIG --> filter by lengh(50-500) for sRNA and that its all. Im not really shure if works well this pipeline. My Problem is what happens with normalization to discard false positive predictions or other considerations.
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You can look in http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3166833/ for example and try to regenerate their results with your pipeline or modify yours according to theirs.